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Journal of Electron Microscopy 51:133-136 (2002)
© 2002 Oxford University Press

Note

Improved method for visualizing coated pits, microfilaments, and microtubules in cryofixed and freeze-substituted plant cells

Takashi Murata1,2, Ichirou Karahara3, Toshiaki Kozuka4,5, Thomas H. Giddings6, Jr, L. Andrew Staehelin6 and Yoshinobu Mineyuki 7,*

1Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan, 3Department of Biology, Faculty of Science, Toyama University, Gofuku, Toyama 930-8555, Japan, 4Department of Mathematical and Life Sciences and 7Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima 739-8526, Japan and6Department of Molecular Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309-0347, USA
Present address: 2National Institute for Basic Biology, Graduate University of Advanced Studies, National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan

*To whom correspondence should be addressed. E-mail: mineyuk{at}hiroshima-u.ac.jp

We have optimized the conditions for visualizing microfilaments, microtubules, and coated pits in the cortical cytoplasm of high-pressure frozen and freeze-substituted plant cells. In both tobacco root tips and onion cotyledons, individual microfilaments and the supramolecular structure of coated pits can be seen clearly in freeze-substituted samples treated with OsO4 at 40°C followed by 5% uranyl acetate. Treatment with uranyl acetate alone resulted in poorly stained cytoplasmic organelles, whereas microfilaments were difficult to discern in specimen treated with OsO4 alone. The combination of a 40°C OsO4 staining step followed by staining with uranyl acetate at 4°C should prove useful for more detailed plant cytoskeletal/membrane studies in the future.

Keywords     coated pit, high-pressure freezing, microfilament, microtubule, osmium tetroxide, uranyl acetate


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